In recent years, development of nanometer-sized structures has received much attention for various molecular biological applications. Gold is probably the most suited element because it exhibits a high chemical stability (noble metal), is characterized by its ability to strongly absorb the visible light at definite wavelengths and is intrinsically not toxic. The thiol (R—SH) modified oligonucleotides serve as attractive tools with a vast number of potential applications in the field of nucleic acid chemistry such as it enables covalent attachment of variety of ligands that contain (a) α,β-unsaturated ketone; (b) maleimide; (c) other Michael acceptor groups; or (d) cysteines in proteins to make disulfide bonds. In addition to this, thiol has a strong specific interaction with gold surface to form reversible covalent bond with gold.
The reactive thiol group can be introduced into oligonucleotides by incorporating sulfide-modified phosphoramidite monomers during oligonucleotide synthesis. Generally, two different types of sulfide modified monomers viz disulfide strategy [Jones, D. S., Hachmann, J. P., Conrad, M. J., Coutts, S., Livingston, D. A. U.S. Pat. No. 5,391,785, 1995] or S-trityl protection [Connolly, B. A.; Rider, P. Nucleic Acids Res. 1985 13, 4485] are very popular to achieve this. Reactive thiol group from the disulfide is generated by treating oligonucleotides with reducing agent such as dithiothreitol (DTT). Whereas, in the other S-trityl strategy, it is generated by cleaving trityl group by silver nitrate. However, this strategy has clear disadvantage of elaborate cleavage process of trityl group with the silver nitrate, which results in relatively poor yields of the final oligonucleotide. Hence, disulfide modified phosphoramidites serve as superior probes for generating thiol groups. The most popular disulfide probes are with the general formula DMT-O—R—S—S—R—O—P(CE)(NiPr2), where R being C3 or C6 spacer arm [Jones, D. S., Hachmann, J. P., Conrad, M. J., Coutts, S., Livingston, D. A. U.S. Pat. No. 5,391,785, 1995]. Because of its interesting properties, we therefore carried out detailed investigation to develop a new synthetic and purification method which gives acyclic disulfide phosphoramidite (where R is C3 spacer) in a high purity for commercial, research and development. Our optimized synthetic protocol is reproducible, suitable for multi gram scale and yields target phosphoramidite in high purity by 31P NMR (>94%). [Srivastava, S. C.; Thatikonda, S. K.; Srivastav, S. K. Shukla, P. U.S. Patent Application No. 2012/000103, 2012].
Nuzzo and Allara have discovered that reactive thiol group adsorb on gold surface and forms ordered mono layers. [Nuzzo, R. G., Allara, D. L. Jour. Am. Chem. Soc. 1983, 105, 4481]. After this, oligonucleotides with thiol group are very much used to generate self-assembled monolayers (SAMs) on the gold surfaces. Although different molecules can be immobilized (silanes, carboxylic acids, pyridines, sulphites and thiols) on different surfaces (gold, silver, platinum, copper, mercury and glass), chemisorption of thiols on gold is a common and simple procedure to immobilize probes on a surface. DNA functionalized gold nanoparticles have since become widely used building blocks in key nucleic acid based assembly strategies and serve as unique probes for recognizing specific sequences in DNA segments [Storhoff, J. J., Elghanian, R., Mucic, R. C., Mirkin, C. A., and Letsinger, R. L. J. Am. Chem. Soc. 1998 120, 1959] as a building blocks for assembling novel structures and materials [Mucic, R. C., Storhoff, J. J., Mirkin, C. A., Letsinger, R. L. J. Am. Chem. Soc. 1998 120, 12674] and bio diagnostics and nano technology based therapeutics [Merkins, C. A., Letsinger, R. L., Mucic, R. C., Storhoff, J. J. Nature, 1996, 382, 607; Hurst, S. J., Hill, H. D., Mirkin, C. A. J. Am. Chem. Soc. 2008, 130, 12192]. It's been proven that formation of these monolayers is influenced by several factors such as temperature, solvent, buffer concentration, chain length of the adsorbate, cleanliness of the substrate, and rate of reaction with the surface and the reversibility of adsorption of the components of the monolayer. These applications depend on the reversible association of gold and sulfur bond between the attached oligonucleotide and nano particle.
The oligonucleotides attached with single thiol group are unstable during the washing steps and formation of stable attachment of oligonucleotides is very important property for its success in applications such as for DNA chip technology. The covalent bond between gold and sulfur is in the order of magnitude from 30 to 40 K cal/mol, which is relatively weak in order to anchor a biopolymer onto a surface. [Dubois L. H., Zegarski B. R., Nuzzo R. G. Proc. Nati. Acad. Sci. USA 1987 84 4739; Liepold, P., Kratzmüller, T., Persike, N., Bandilla, M., Hinz, M., Wieder, 15H., Hillebrandt, H., Ferrer, E., Hartwich, G. Anal Bioanal Chem, 2008, 391, 1759-1772]. It has been reported that oligonucleotides that are conjugated with mono functional thiol group are slowly lost at higher temperatures and also in the presence of high salt concentration buffers [Li, Z., Jin, R., Mirkin, C. A., Letsinger, R. L. Nucleic Acids Res. 2002, 30, 1558]. The stability studies by Letsinger et. al. on SAMs of oligonucleotides that are conjugated to gold surface by mono thiol group revealed that these are completely displaced by treating with the buffers containing DTT [Letsinger, R. L., Elghanian, R., Viswanadham, G., Mirkin, C. A. Bioconj. Chem. 2000, 11, 289]. This feature limits applications of these probes in solutions containing thiols such as a PCR solution that has DTT as a stabilizer for the polymerase enzyme.
So there is strong need to develop novel disulfide compounds that are capable of forming stable SAMs of oligonucleotides for wider biological applications. One can anticipate that stability of mono layers could be increased by multiple numbers of gold-sulfur bonds per oligonucleotides. There have been few reports [Letsinger, R. L., Elghanian, R., Viswanadham, G., Mirkin, C. A. Bioconj. Chem. 2000, 11, 289-291; Hartwich, G., Frischmann, P., Ferrer, E., U.S. Pat. No. 7,601,848, 2002; Seliger, H., Prokein, T. U.S. Patent No 2005/0059728, 2004] that has introduced novel thiol modifiers which can generate multiple thiol groups per oligonucleotide. Its been proved that SAMs produced by these modifications are much more stable than corresponding SAMs generated by mono thiol modifier in buffers containing DTT [Letsinger, R. L., Elghanian, R., Viswanadham, G., Mirkin, C. A. Bioconj. Chem. 2000, 11, 289]. However the DMT group attached to the side chain in the disclosed art is attached to a secondary hydroxyl, which can effect the quality of oligonucleotides attached to solid surfaces. The present invention utilizes nucleoside 5′-DMT group for oligonucleotide growth band thereby synthesis of high quality dithiolane oligonucleotides.
Chart 1:
Chemical structures of N2-Guanosine (O6-protected) functionalized dithiolane phosphoramidites 1, solid supports 2 and previously disclosed dithiolane phosphoramidites 3 and their solid supports 4.

Previously, we have described five membered disulfide (dithiolane) based anchoring group 3 (Chart 1) for the introduction of two thiol groups [Srivastava, S. C., Thatikondra, S. K., Srivastav, S. K., Shukla, P. K., Srivastava, A., U.S. Provisional Application is 61/795,851, filed Oct. 27, 2012; U.S. Non-Provisional Application Ser. No. 14/065,385, filed Oct. 28, 2013.]. This dithiolane modification offered primary hydroxyl group to attach DMT group for the purpose of oligonucleotide synthesis, offers high quality oligonucleotide, and can potentially afford gold-oligonucleotide conjugates that exhibit greater stability.